Tumor‐Targeted Injectable Double‐Network Hydrogel for Prevention of Breast Cancer Recurrence and Wound Infection via Synergistic Photothermal and Brachytherapy

Abstract The high locoregional recurrence rate and potential wound infection in breast cancer after surgery pose enormous risks to patient survival. In this study, a polyethylene glycol acrylate (PEGDA)‐alginate double‐network nanocomposite hydrogel (GPA) embedded with 125I‐labeled RGDY peptide‐modified gold nanorods (125I‐GNR‐RGDY) is fabricated. The double‐network hydrogel is formed by injection of GPA precursor solutions into the cavity of resected cancerous breasts of mice where gelation occurred rapidly. The enhanced temperature‐induced PEGDA polymerization driven by near‐infrared light irradiation, and then, the second polymer network is crosslinked between alginate and endogenous Ca2+ around the tumor. The double‐network hydrogel possesses a dense polymer network and tightly fixes 125I‐GNR‐RGDY, which exhibit superior persistent photothermal and radioactive effects. Hyperthermia induced by photothermal therapy can inhibit self‐repair of damaged DNA and promote blood circulation to improve the hypoxic microenvironment, which can synergistically enhance the therapeutic efficacy of brachytherapy and simultaneously eliminate pathogenic bacteria. Notably, this nanocomposite hydrogel facilitates antibacterial activity to prevent potential wound infection and is tracked by single‐photon emission computerized tomography imaging owing to isotope labeling of loaded 125I‐GNR‐RGDY. The combination of photothermal therapy and brachytherapy has enabled the possibility of proposing a novel postoperative adjuvant strategy for preventing tumor recurrence and wound infection.


Experimental Section
Characterization: The morphology of the GNR was observed by transmission electron microscopy (TEM) (JEM-2100, Japan). Scanning electron microscopy (SEM) was conducted at an accelerate voltage of 5 kV using a FEI Quanta S-4800 microscope (Japan). The hydrogel samples were flash frozen in liquid nitrogen for 5 min and immediately lyophilized for 2 days to remove water. Au coating of samples for imaging was carried out by sputtering for 45 s. UV-Vis spectrophotometry measurements were performed on TU-1810 UV-Vis spectrophotometer with a wavelength range from 400 to 1000 nm. In vivo isotope imaging was carried out by using SPECT-CT system (Triumph X-SPECT2h/X-OCT).
Cytotoxicity Assay: CCK-8 was used to evaluate the cell cytotoxicity of the nanoparticles and polymers used in this experiment. Before detection, the sample disks (5 mm in diameter) were cut from hydrogel films, immersed in 75% ethanol for 2 h for sterilization and then washed with PBS overnight. Subsequently, the PA and GPA samples were placed in a 96-well plate, and fixed on the bottom of the wells.
Fibroblasts L929 suspension (100 μL) was added into 96-well microplates, with 5000 cells immersed in the complete growth medium per well, cultivated in a humidified 5% CO 2 atmosphere at 37 C for 24 h to allow cells to attach. Then, CCK-8 solution was added to 96-well plates at 10 µL per well and incubated for 3 h. The resulting solutions were analysed at 480 nm on a plate reader (BIO-TEK instruments Inc EL311S, America). This process was repeated for 8 times in parallel. The results were expressed as the relative cell viability (%) with respect to blank group only with culture medium. The cell viability in each well was calculated from the obtained values as a percentage of control wells. The results were presented as a mean and standard deviation obtained from each samples.

In Vitro Antibacterial Evaluation of the Hydrogels: Escherichia coli (E. coil), Staphylococcus aureus (S. aureus) and Methicillin-resistant Staphylococcus aureus
(MRSA) were employed to test the antibacterial activity of the hydrogel. In brief, bacteria were divided into four groups with different treatments: (1) PBS; (2) methicillin; (3) GPA hydrogel; (4) GPA hydrogel with NIR irradiation. The prepared hydrogel samples were added into 48-well plate and then 10 μL bacterial suspension (in PBS, 10 6 CFU/mL) was added onto the surface of hydrogel disks. Next, this 48-well plate was put into incubator at 37 °C for 2 h in a relative humidified atmosphere. At the end of that time, 1 mL of sterilized PBS was added to each well to re-suspend any bacterial survivors. As the control, 10 μL of bacterial suspension in sterilized PBS (10 6 CFU/mL) was added in 1 mL PBS to obtain a homogeneous solution. After incubation for 18-24 h at 37 °C, the colony-forming units (CFU) on the Petri dish were counted. Tests were repeated three times for each group and the killing ratios of bacteria by hydrogels were measured.
Hemolysis Assay: Fresh blood was obtained from SD rats, whose erythrocytes were separated by centrifugation at 3000 rpm for 10 minutes, washed three times with saline solution, and finally diluted with erythrocyte stock (100 μL, 5%). The samples of PA and GPA was added to the solution, respectively, and incubated for 1 h at 37 °C.
Then the samples were centrifuged at 1000 rpm for 5 minutes and the supernatants were collected for further detection. Hemolytic activity was determined by OD 545nm using a multifunctional microplate reader. Red blood cells (RBC) in saline solution was a negative control and deionized water was a positive control. The hemolysis percentage (Hemolysis %) was calculated from the following equation: where A S is the absorbance of the samples, Ais the absorbance of the negative control and A + is the absorbance of the positive control. Pathology Analysis: On the 28th day after treatment, the mice were euthanized, and the representative organs including heart, liver, spleen, lung, and kidney were excised, fixed with a 4% paraformaldehyde solution overnight, embedded in paraffin, sliced into 8 μm thick sections, and stained with H&E. Ultimately, the slices were observed and photographed using an optical microscope (Leica, DMI 3000 B) for the pathology analysis to evaluate the biosafety of the hydrogel during the treatment.